| Autistic spectrum disorder (ASD) refers to a group of neurodevelopmental disorders. Typically diagnosed before three years old, autistic subjects usually present with significant language delays, social and communication impairments, and abnormal repetitive and stereotypic behaviors. Studies in Asia, Europe and North America have identified ASD individuals with an approximate prevalence of 6/1,000 to over 10/1,000. There's no accurate epidemiological data regarding the prevelance in China, but it was estimated there are over one billion autism patients in China.It's now generally accepted that both genetic and environmental factors attribute to ASD, among which prenatal factors are the most important ones. Disturbances during early organogenesis (i.e. Day 20 to Day 40 after fertilization) lead to significantly increased risk to develop ASD in the postnatal life.Ras/Raf/ERKl/2 pathway plays important roles in the genesis of neural progenitors and synaptic transduction, therefore is critical to cognition, memory and learning abilities. In addition, Ras/Raf/ERK1/2 pathway is reported to involve in neuron death. Recently, it was demonstrated that Ras/Raf/ERK1/2 pathway underlies several mental retardations, but its relationship with autism remains undetermined. Our previous studies revealed abnormal decrease of anti-apoptotic protein Bc12 and elevated level of pro-apoptotic protein p53 in autistic cortex and cerebellum, indicating abnormal neuron death in autism. Another recent study related deletion of ERK1 gene to the pathogenesis of autism. Based on these evidences, we proposed that impaired Ras/Raf/ERK1/2 pathway is associated with autism. To testify our hypothesis, we intended to measure the expression levels and activities of major members within Ras/Raf/ERKl/2 pathway using both human and mice brain samples. Then we test the behavior changes of neurons by correcting the abnormal Ras/Raf/ERK1/2 pathway, hoping to provide preliminary information regarding autism treatment.CHAPTER 1 UP-REGULATION OF RAS/RAF/ERK1/2 PATHWAY IN AUTISTIC BRAINSOBJECTIVERas/Raf/ERK1/2 pathway plays important roles in the genesis of neural genitors and neural crest, as well as synaptic transduction, therefore is critical to cognition, memory and learning ability. Cumulative data suggested abnormal Ras/Raf/ERK1/2 pathway underlies several mental retardations, based on which we hypothesized that this pathway is also related to the pathogenesis of autism. To test this hypothesis, we measured the expression levels and activities of the major members within this pathway.METHODS1. Study human subjectsFrozen human brain tissues of six autistic subjects (mean age (8.3±3.8) years) and six age-matched normal subjects (mean age (8±3.7) years) were obtained from the NICHD Brain and Tissue Bank for Developmental Disorders. Donors with autism fit the diagnostic criteria of the Diagnostic and Statistical Manual-IV, as confirmed by the Autism Diagnostic Interview-Revised. Participants were excluded from the study if they had a diagnosis of fragile X syndrome, epileptic seizures, obsessive-compulsive disorder, affective disorders, or any additional psychiatric or neurological diagnoses.2. Preparation of brain tissue homogenateThe frozen frontal cortex and cerebellum tissues were homogenized seperately in cold buffer (10% w/v) containing 50 mM Tris-HCl (pH 7.4),8.5% sucrose,2 mM EDTA,10 mMβ-mercaptoethanol and a protease inhibitors cocktail respectively. The protein concentration was assayed by the Bradford method. 3. Western Blot AnalysisBrain homogenate samples in SDS sample buffer (20% glycerol,100 mM Tris, pH 6.8,0.05% Bromophenol blue (w/v),2.5% SDS (w/v),250mM DTT) were denatured by heating at 100℃for five minutes. Twenty to Sixty micrograms of protein per lane per subject was loaded onto a 10% acryl-bisacrylamide gel and electrophoresed for 2 hours at 110V at room temperature. The proteins were electroblotted onto a PVDF membrane for 1 hour at 100V at room temperature. Protein blots were then blocked with 5% non-fat milk in phosphate buffered saline (PBS) with 0.1% Tween-20 (PBST). After blocking, the blots were incubated with primary antibody overnight at 4℃followed by a secondary antibody incubation for 1 hour at room temperature (goat ant-mouse IgG or goat anti-rabbit IgG, HRP conjugated,1:5000, Sigma). After three washes in PBST (10 minutes each time), the blots were exposed to Hyper film ECL. Sample densities were analyzed with Image J software. The densities of the protein expression bands, as well as theβ-actin expression bands were quantified with background subtraction.4. Immunohistochemistry6μm paraffin sections were deparaffinized with xylene (2X), ethanol of 100% (2X),80%,50%,25% concentration and washed in TBS,5 minutes each time. The sections were then incubated with primary antibodies overnight at 4℃. After washing in TBS for 5 minutes, the sections were further incubated with secondary antibody (biotinylated horse anti-mouse IgG, or biotinylated horse anti-rabbit IgG, VectaStain Elite ABC Kit, Vector Lab) for 30 minutes at room temperature, followed by incubation in Avidin-biotinylated peroxidase (VectaStain Elite ABC Kit) for 45 minutes at room temperature and in 0.0125g DAB/25 ml 0.05M TBS/1 drop 30% H2O2 for 10 minutes at room temperature. All sections were washed in sequence with TBS,25%,50%,80%,100% ethanol (2X) and xylene (2X) before mounting for viewing under the microscope.5. Imaging and data qualificationImmunostaining images were visualized using a laser scanning confocal microscope to obtain clear pictures (Nikon Eclipse 90i,10 x 40 maglification, IBR-Microscopy Shared Research Facility). Image J software was used to calculate area and immunostaing density.6. Statistical analysisMeans, standard deviations and standard errors of the mean were determined in sets of autistic subjects versus age-matched control subjects. Unpaired t test was used to compare each parameter. P< 0.05 is considered statistically significant.RESULTS1. Western blot studies were conducted to examine RAS protein expression in the frontal cortex and cerebellum of autistic subjects and normal controls. The bands representing the RAS protein expression were stronger in frontal cortex of autistic subjects as compared with the controls. Quantitative analysis showed that the mean value of RAS expression increased 17.91% in frontal cortex of autistic subjects as compared with the controls (t=-2.784, P=0.019), while no significant difference was detected in cerebellum (t=-1.758, P=0.109).2. C-Raf phosphorylation/activation was significantly increased by a mean value of 64.10%(t=-3.662, P=0.006) in the cortex and 42.20%(t=-2.677, P=0.042) in autistic cerebellum as compared with controls, although C-Raf protein expression remained unchanged (P>0.05). We did not detect significant alterations in both the protein expression and phosphorylation of A-Raf and B-Raf in autistic brains as compared with controls. These results suggest that Raf kinases are differently regulated in the cortex and cerebellum.3. Our results showed that the phosphorylation/activation of MEK1/2 were significantly increased by a mean value of 19.63% in the frontal cortex of autistic subjects as compared with controls (t=-6.315, P<0.001), while no significant difference of the total protein expression of MEK1/2 was detected (t=1.590, P=0.143). The results of immunohistostaining using p-MEK1/2 further comfirmed these results, in that significant more positive neurons in autistic brains were detected(t=5.320, P<0.001). However, the phosphorylation of MEK1/2 in autistic cerebellum remained unchanged (P>0.05).4. Compared with normal controls, ERK1/2 expression was significantly increased by a mean value of 56.54% in autistic cortex (t=-2.288, P=0.045). We however did not detect significant alterations in the phosphorylation of ERK1/2 using either Western blot or immunostaining, which was probably due to the low expression level of this protein.CONCLUSIONCompared with normal subjects, significant up-regulation of Ras/Raf/ERK1/2 pathway was detected in autistic brains, which may underlies the pathogenesis of autism.CHAPTER 2 UP-REGULATIONS OF RAS/RAF/ERK1/2 PATHWAY IN BTBR MICE BRAINSOBJECTIVEBehavior tests indicated that BTBR mice exhibited all the three core symptoms of autism, therefore is considered as an ideal animal model for autism research. Growing evidences suggested that mTOR pathway together with Ras/Raf/ERK1/2 pathway played important roles in the pathogenesis of mental retardations. In addition, our previous experiments found up-regulations of Ras/Raf/ERK1/2 pathway in autistic subjects. We thus intend to explore the relationship between autism and Ras/Raf/ERK1/2 and mTOR pathway using BTBR mice as animal model.METHODS1. Brain sample preparationSix female BTBR T+tfJ (BTBR) and six B6 were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Mice were housed for 24 hours with ad lib food and water to ease the stress before sacrifice. Then mice were rapidly sacrificed with cervical dislocation for removal of the brains.All procedures were conducted in compliance with the NIH Guidelines for the Care and Use of Laboratory Animals and approved by the New York State Institute for Basic Research Institutional Animal Care and Use Committee.2. Preparation of brain tissue homogenateThe frontal cortex and cerebellum were dissected respectively. The frozen frontal cortex and cerebellum tissues were homogenized (10% w/v) in cold buffer containing 50 mM Tris-HCl (pH 7.4),8.5% sucrose,2 mM EDTA,10 mMβ-mercaptoethanol and a protease inhibitor cocktail respectively. The protein concentrations were assayed by the Bradford method.3. Western Blot AnalysisSamples underwent electrophoresis as described previously. Sample densities were analyzed with Image J software. The densities of the protein expression bands, as well as theβ-actin expression bands were quantified with background subtraction.4. Immunohistochemistry6μm paraffin sections underwent immunostaining as as described previously.5. Imaging and data qualificationImmunostaining images were visualized using a laser scanning confocal microscope to obtain clear pictures (Nikon Eclipse 90i,10 x 40 maglification, IBR-Microscopy Shared Research Facility). Image J software was used to calculate area and immunostaing density.6. Statistical analysisMeans, standard deviations and standard errors of the mean were determined in sets of BTBR mice versus B6 controls. The unpaired t test was used to compare each parameter. P< 0.05 is considered statistically significant.RESULTS1. Western blot studies were conducted to examine RAS protein expression in the frontal cortex and cerebellum of six BTBR mice and six B6 control mice. The bands representing the RAS protein expression were stronger in frontal cortex as well as in the cerebellum of BTBR mice group as compared with the controls. Quantitative analysis showed that the mean value of RAS expression increased by 134.34%(t=-3.642, P=0.005) in frontal cortex and 73.79%(t=-4.482, P=0.001) in cerebellum of BTBR mice, as compared with the controls.2. Our results showed that the phosphorylation/activation of A-Raf, B-Raf and C-Raf were significantly increased by 94.82%(t=-5.441, P=0.002),167.95% (t=-7.693, P=0.001) and 40.20% (t=-2.357, P=0.040) respectively in the frontal cortex of BTBR mice as compared with B6 mice, while the total protein expression of A-Raf (t=1.297, P=0.233), B-Raf (t=-1.464, P=0.174) and C-Raf (t=1.182, P=0.264) remained unchanged. The results of immunohistostaining using p-C-Raf was consistent with these results, in that significant more positive neurons in autistic brains were detected (t=2.877, P=0.007).3. C-Raf phosphorylation/activation was significantly increased by 58.27% in the cerebellum of BTBR mice as compared with control B6 mice (t=-4.532, P=0.001), although C-Raf protein expression remained unchanged (t=-0.521, P=0.614). We did not detect significant alterations in both the protein expression and phosphorylation of A-Raf and B-Raf in the cerebellum of BTBR mice as compared with B6 mice. These results suggest that Raf kinases are differently regulated in the cortex and cerebellum of BTBR mice.4. We detected that MEK1/2 phosphorylation/activation was dramatically increased in the frontal cortex of BTBR mice by 385.40%(t=-14.547, P<0.001) as compared with the B6 mice, while total protein expression remained unchanged (t=0.317, P=0.758). To further confirm these results, we examined MEK1/2 phosphorylation in the frontal cotex of BTBR mice with immunohistochemistry studies. Our results further demonstrated that phospho-MEK1/2 is markedly increased in the cortical neural cells of BTBR mice as compared with control B6 mice, which is consistent with the findings from Western blot studies(t=10.425, P<0.001). We also examined the protein expression and phosphorylation of MEK1/2 in the cerebellum of BTBR mice. However, we failed to find significant changes in both the protein expression (t=0.007, P=0.995) and phosphorylation of MEK1/2 (t=2.189, P=0.053) in BTBR as compared with B6 (P>0.05).5. We observed that ERK1/2 phosphorylation/activation was dramatically increased in the frontal cortex of BTBR mice by a mean value of 54.92% compared with the B6 mice (t=-7.842, P<0.001), while total protein expression remained unchanged (t=-0.611, P=0.555). To further confirm these results, we examined ERK1/2 phosphorylation in the frontal cotex of BTBR mice with immunohistochemistry studies. Our results further demonstrated that phospho-ERK1/2 markedly increased in the cortical neurons of BTBR mice as compared with control B6 mice, which is consistent with the findings from Western blot studies (t=-12.041, P<0.001). However, we did not find significant changes in both the protein expression (t=1.428, P=0.184) and phosphorylation (t=0.255, P=0.804) of ERK1/2 in BTBR cerebellum as compared with B6 (P>0.05).6. We further detected significant increase of p-Akt, p-mTOR and p-p70 S6, which were the three main effectors in mTOR pathway. Compared with control group, the mean increase values of p-Akt, p-mTOR and p-p70 S6 were 32.91%(t=-2.802, P=0.035),112.44%(t=-10.495, P<0.001) and 41.5%(t=-3.226, P=0.022), respectively, while the total expression levels of Akt (t=1.924,P=0.101), mTOR (t=1.198, P=0.259) and p70 S6 (t=-0.689, P=0.514) remained unchanged as compared with B6. And we failed to find any significant change in both the protein expression and phosphorylation of the three proteins in BTBR cerebellum as compared with B6 (P>0.05).CONCLUSIONUp-regulation of Ras/Raf/ERK1/2 pathway was detected in both cortex and cerebellum of BTBR mice, which is consistent with the previous results from human bain samples. These results further support our hypothesis that Ras/Raf/ERK1/2 pathway may underlie the pathogenesis of autism. In addition, up-regulation of mTOR pathway was detected in BTBR mice brains, indicating it may play a role in the pathogenesis of autism, too. But the interaction between the two pathways requires further exploration.CHAPTER 3 MEK INHIBITOR U0126 AND MTOR INHIBITOR RAPAMYCIN PARTLY RESCUE THE ABNORMAL BEHAVIORS OF CORTICAL NEURONS OF BTBR MICEOBJECTIVEWe have shown that the up-regulations of Ras/Raf/ERK1/2 and mTOR pathways in BTBR mice brain, which may underlie the pathogenesis of autism. In this part we employed two inhibitors, MEK inhibitor U0126 and mTOR inhibitor rapamycin, to specifically inhibit over expression of these two pathways. And we further explored the possible changes of neuron behaviors after the inhibitions, providing evidence for autism therapies.METHODS1. Cell cultureCortical cells were prepared from C57BL/6J and BTBR fetuses (sacrificed at 16th embryonic day). Briefly, the entire cortex was removed, and single cell suspension was prepared by trypsinization and trituration in 0.125% trypsin in culture medium (MEM). After centrifuge and remove the supernatant, cells were re-suspended in MEM, supplemented with 10% fetal bovine serum (FBS) and 100 U/ml both penicillin and streptomycin. Cells were seeded into PDL-coated dishes and incubated at 37℃C in a moist chamber with 5% CO2. After 24 hours in vitro the medium was replaced with serum-free NeuroBasal medium containing 2% B27 supplement,0.5mM GlutaMax and 100U/ml both penicillin and streptomycin in the medium.2. Cell treatmentsAfter cultured for 7-10 days, cells underwent the following treatments before used for experiment. For Western blot, cells were treated with either MEK inhibitor U0126 or mTOR inhibitor rapamycin or both at different concentrations for one hour before the cells were harvested. For adhesion and migration experiments, cells were treated with either U0126 (10μM) or rapamycin (0.1 nM) or both at indicated concentrations for one hour before they were trysinized. For Dil labeling, cells were treated with U0126 (10μM) for three successive days with renewal of the medium every 24 hours to avoid accumulations of cell metabolites or drugs.3. Western blotCultures were harvested and homogenized (10% w/v) in ice-cold buffer containing 50 mM Tris-HCl (pH 7.4),8.5% sucrose,2 mM EDTA,10 mMβ-mercaptoethanol and a protease inhibitors cocktail (Sigma-Aldrich) respectively. The protein concentration was assayed by the Bradford method. Samples underwent electrophoresis as described previously. The densities of the protein expression bands, as well as theβ-actin expression bands were quantified with background subtraction using ImageJ software. All procedures repeated at least three times.4. Cell adhesion assay5000 cortical cells from C57BL/6J or BTBR mice were plated per well in 96 well tissue culture plates coated with recombinant ICAM-1 (R&D Systems) at the final concentration of 10μg/ml. After 1.5 hours of attachment, unattached cells were removed by aspiration, the adherent cells were quantitated by the colorimetric aqueous MTS assay (CellTiter 96 AQueous One Solution kit, Promega).5. Cell migration assayCortical cells were labeled with fluorescent Calcein AM (BD Biosciences) at the final concentration of 2.5μM.5000 labeled cells were plated in 0.8 ml MEM+1% FBS in each well of 24 well chambers adapted for the HTS Fluoroblock (BD Falcon) apparatus. After 3 hours,5% FBS was added to the lower chamber medium to establish a 1-5% serum gradient, the migration of cells from the upper to lower chambers was quantitated at 2 hours using a microfluorimetric plate reader (CytoFluor 4000, MTX Lab Systems).6. ImmunofluorescenceCortical cells were fixed in 4% formaldehyde for 15 min and blocked with 3% goat serum/0.3% Triton X-100 in PBS and incubated with anti-synaptophysin polyclonal antibody (synaptophysin,1:1000, Cell Signaling Technology), anti-VGLUT1 monoclonal antibody (1:500, Millipore), and anti-VGAT polyclonal antibody (1:500, Millipore) overnight at 4℃, followed by incubation with Alexa Fluor 555 anti-rabbit and Alexa Fluor 488 anti-mouse IgG (1:1000, Invitrogen) for 1.5 hours at room temperature. Cultures were mounted on glass slides with ProLong Gold antifade reagent (Invitrogen), and then imaged using Nikon eclipse 90i confocal laser scanning microscope. The expressions of synaptic proteins were quantified with ImageJ software.7. DiI labelingAfter treated with U1026 (10μM) for three successive days, cultures were labeled with Dil dye. Briefly, cells were fixed in 4% formaldehyde for 15 min and incubated with Vybrant-DiI cell-labeling solution (1:200, Invitrogen) for 25 min at 37℃. Cultures were washed in warm PBS, followed by incubation in PBS at 4℃for 24 hours to allow dye diffusion within membranes before being mounted on glass slides with ProLong Gold antifade reagent (Invitrogen). Images were acquired using Nikon Eclipse E800 microscope. Mature dendritic spines appear mushroom- or stubby-shaped morphology, while immature spines appear thin morphology.8. Statistical analysisStatistical analysis was conducted using SPSS 13.0 software. All graphic data are shown as means±SED. Synapse comparisons were analyzed by unpaired t test. Cells treated with two inhibitors were analyzed by univariate test. All the other data were first statistically probed by one-way ANOVA, then data with significant overall difference among the groups were further analyzed for between-group differences with one-way ANOVA. Significance was accepted at P<0.05.RESULTS1. Compared with B6 cortical neurons, the fluorescence tensity of synaptophysin, VGLUT1 and VGAT in BTBR cortical neurons are 32.06%(t=10.536, P<0.001), 23.98%(t=7.525, P<0.001) and 16.18%(t=5.982, P<0.001) lower, respectively. The ratio of VGLUT1/VGAT of BTBR cortical neurons was 11.16%(t=-2.444, P=0.015) lower than B6 cortical neurons.2. U0126 inhibited the activation of Ras/Raf/ERK1/2 pathway in a dose-dependent manner.(1) There are significant difference within B6 groups as to the level of p-ERK (F=12.159, P=0.002). Intra-B6 group, comparing with control, the density of p-ERK decreased 10.55%(P=0.036),9.81%(P=0.047) and 25.07%(P<0.001) in U0126 10μM,20μM and 30μM groups, respectively.(2) There are significant difference within BTBR groups as to the level of p-ERK (F=92.189, P<0.001). Intra-BTBR group, comparing with control, the density of p-ERK decreased 30.14%(P<0.001),36.40%(P<0.001) and 66.00% (P<0.001) in U0126 10μM,20μM and 30μM groups, respectively.(3) Inter-groups, the density of p-ERK in BTBR control group increased 14.84% compared with B6 control group (t=-3.143, P=0.035). The density of p-ERK decreased 10.31% in BTBR U0126 10μM group compared with B6 U0126 10μM group (t=5.335, P=0.006). The density of p-ERK decreased 19.40% in BTBR U0126 20μM group compared with B6 U0126 20μM group (t=5.114, P=0.028). The density of p-ERK decreased 32.54% in BTBR U0126 30μM group compared with B6 U0126 30μM group (t=4.529, P=0.011).(4) No significance were detected between groups as to the levels of ERK protein (B6 group F=0.223, P=0.878, BTBR group F=0.403, P=0.755).3. Rapamycin inhibited the activation of mTOR pathway in a dose-dependent manner.(1) There are significant difference within B6 groups as to the level of p-p70 S6 (F=83.488, P<0.001). Intra-B6 group, comparing with control, the density of p-p70 S6 decreased 20.11%(P=0.007),39.97%(P<0.001) and 84.34% (P<0.001) in rapamycin 0.1nM, 1nM and 10nM groups, respectively.(2) There are significant difference within BTBR groups as to the level of p-p70 S6 (F=122.582, P<0.001). Intra-BTBR group, comparing with control, the density of p-p70 S6 decreased 31.74%(P<0.001),40.03%(P<0.001) and 54.98%(P<0.001) in rapamycin 0.1nM, 1nM and 10nM groups, respectively.(3) Inter-groups, the density of p-p70 S6 in BTBR control group decreased 33.74% compared with B6 control group (t=6.853, P=0.002). The density of p-p70 S6 in BTBR rapamycin 0.1nM group decreased 43.38% compared with B6 rapamycin 0.1nM group (t=18.555, P<0.001). The density of p-p70 S6 in BTBR rapamycin 1nM group decreased 33.80% compared with B6 rapamycin 1nM group (t=4.641, P=0.035). The density of p-p70 S6 in BTBR rapamycin lOnM group increased 90.48% compared with B6 rapamycin lOnM group (t=-2.944, P=0.042).(4) No significance were detected between groups as to the levels of p70 S6 (B6 group F=0.531, P=0.674. BTBR group F=1.573, P=0.270).4. Combination effects on B6 cortical neurons using U0126 and rapamycin.(1) Intra-B6 group, significant decrease of the p-ERK levels in B6 neurons were detected after treated with either U0126 (F=5.666, P=0.045) or rapamycin (F=57.996, P<0.001) or both (F=18.050, P=0.003), comparing with controls.(2) Intra-B6 group, significant increase of the adhesion abilities of B6 neurons were detected after treated with either rapamycin (F=1407.240, P<0.001) or rapamycin and U0126 (F=6.116, P=0.039), but not U0126 alone (F=0.058, P=0.816), comparing with controls.(3) No significance were detected between groups as to the levels of ERK or p70 S6 proteins (P>0.05).5. Combination effects on BTBR cortical neurons using U0126 and rapamycin.(1) Significant decrease of the p-ERK levels in BTBR neurons were detected after treated with either U0126 (F=819.866, P<0.001) or rapamycin (F=951.046, P<0.001) or both (F=727.075, P<0.001), comparing with controls.(2) Significant decrease of the p-ERK levels in BTBR neurons were detected after treated with either U0126 (F=74.390, P<0.001) or rapamycin (F=11038.788, P<0.001) or both (F=33.265, P<0.001), comparing with controls.(3) No significance were detected between groups as to the levels of ERK or p70 S6 proteins (P>0.05).6. Cell adhesion assay.(1) Intra-B6 group, significant increase of the adhesion abilities of B6 neurons were detected after treated with either U0126 (F=6.296, P=0.023) or rapamycin and U0126 (F=10.114, P=0.006), but not rapamycin alone (F=0.917, P=0.353), comparing with controls.(2) Intra-BTBR group, significant increase of the migration abilities of BTBR neurons were detected after treated with either U0126 (F=10.576, P=0.005) or rapamycin (F=4.920, P=0.041), but not rapamycin alone (F=2.101, P=0.167), comparing with controls.(3) Inter-groups, adhesive cells in BTBR control group decreased 66.57% compared with B6 control group (t=12.704, P<0.001); adhesive cells in BTBR rapamycin group decreased 60.55% compared with B6 rapamycin group (t=9.773, P<0.001); adhesive cells in BTBR U0126 group decreased 57.49% compared with B6 U0126 group (t=6.833, P<0.001); adhesive cells in BTBR rapamycin/U0126 group decreased 55.05% compared with B6 rapamycin/U0126 group (t=8.529, P=0.001).7. Cell migration assay.(1) Intra-B6 group, significant increase of the migration abilities of B6 neurons were detected after treated with either U0126 (F=65.581, P<0.001) or rapamycin (F=6.414, P=0.035) or both (F=8.913, P=0.017), comparing with controls.(2) Intra-BTBR group, significant increase of the migration abilities of BTBR neurons were detected after treated with either U0126 (F=25.525, P=0.001) or rapamycin(F=32.940, P<0.001) or both (F=5.597, P=0.046), comparing with controls.(3) Inter-groups, cell migration ability in BTBR control group decreased 28.88% compared with B6 control group (t=3.217, P=0.032); cell migration ability in BTBR rapamycin group decreased 27.22% compared with B6 rapamycin group (t=3.572, P=0.023); adhesive cells in BTBR U0126 group decreased 46.05% compared with B6 U0126 group (t=10.928, P<0.001); adhesive cells in BTBR rapamycin/U0126 group decreased 37.34% compared with B6 rapamycin/U0126 group (t=8.396, P=0.013).8. Dil staining indicated that the spine density of BTBR neurons was 62.92% fewer than B6 neurons (t=7.006, P<0.001). However, U0126 significantly increased the density of dendrite spines in both B6 and BTBR neurons. Compared with untreated BTBR neurons, the spine density of BTBR neurons increased 251.61% (t=7.894, P<0.001) after treated with U0126 10μM for three successive days. The density of B6 neurons increased 83.58% fter treated with U0126 lOμM for three successive days (t=4.188, P<0.001).CONCLUSIONMEK inhibitor U0126 and mTOR inhibitor rapamycin suppressed the up-regulations of Ras/Raf/ERKl/2 and mTOR pathways in BTBR mice cortical neurons. The abnormal behaviors of BTBR mice cortical neurons were partially rescued by the treatments of U0126 and/or rapamycin, which suggested these inhibitors have potential therapeutic effects for autism.SUMMARY1. Compare with normal subjects, significant up-regulation of Ras/Raf/ERK1/2 pathway was detected in autistic brains, which may underlies the pathogenesis of autism.2. Up-regulations of Ras/Raf/ERK1/2 and mTOR pathways were detected in both cortex and cerebellum of BTBR mice, which is consistent with the previous results from human bain samples. These results further support our hypothesis that Ras/Raf/ERK1/2 and mTOR pathways may underlie the pathogenesis of autism. In addition, up-regulation of mTOR pathway was detected in BTBR mice brains, indicating it may play a role in the pathogenesis of autism.3. MEK inhibitor U0126 and mTOR inhibitor rapamycin suppressed the up-regulations of Ras/Raf/ERK1/2 and mTOR pathways in BTBR mice cortical neurons. The abnormal behaviors of BTBR mice cortical neurons were partially rescued by the treatments of U0126 and/or rapamycin, which suggested these inhibitors have potential therapeutic effects for autism.
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